DESCRIPTION (Verbatim from Applicant's Abstract): Invasive disease caused by Group A streptococci (GAS) continues to be an infrequent but often devastating medical problem in humans. Antibiotics and surgery are the only mainstays of treatment, because our understanding of the pathogenesis of these infections is insufficient to design rational interventions and vaccines. Our research group is studying a signal transduction system in GAS that controls the expression of several genes required for development of invasive skin infection in mice. The streptococcal proteins CsrS and CsrR comprise a two-component regulatory system. CsrS is a membrane-spanning sensor-kinase molecule that presumably phosphorylates CsrR, a DNA-binding regulator. Phosphorylated CsrR binds upstream of specific genes and represses expression of hyaluronic acid capsule, streptolysin S, pyrogenic exotoxin B, streptokinase, and mitogenic factor. Mutation of csrR results in bacteria with enhanced expression of these virulence factors and dramatically more severe skin lesions in mice. Our laboratory will use genetic methods to determine how the CsrRS system functions and whether spontaneous mutations in this locus affect the outcome of human disease. 1) We will analyze the contributions of key CsrR-regulated factors in the development of dermonecrotic skin lesions in order to define which factors are necessary for aggressive disease. 2) We will determine how the phosphorylation of CsrR correlates with the expression of virulence factors at various points in the bacterial growth cycle. Because preliminary data show that csrS mutants persistently repress CsrR-regulated virulence genes, we will study both the kinase and phosphatase activity of CsrS independently to determine how its activity relates to the phosphorylation of CsrR. 3) csrS mutants produce larger, more mucoid colonies when grown in a 5 percent C02 atmosphere instead of air, and the bacteria are more virulent than wild type. We propose to determine the basis for the phenotypic change and to determine whether it accounts for the enhanced virulence. 4) We have observed that spontaneous mutations in csrRS occur often in vivo. When this occurs, there is synergy between the mutant and wild type strains at the site of infection that enhances bacterial growth, local invasion, and bacteremia in mice. We will use strains with two copies of csrRS to evaluate the basis for this synergy in mice in order to avoid the spontaneous emergence of mutants during wild type infections. 5) Finally, we propose to study human isolates from patients with invasive and non-invasive streptococcal infections to determine whether subpopulations of csrRS mutant bacteria are a common feature of these life-threatening diseases. If so, this observation will have major impact on the future analyses of human cases.